Gene cluster involved in biosynthesis of isopentenyl diphosphate in the non-mevalonate pathway of Hevea brasiliensis

ABSTRACT

According to this invention, a gene cluster involved in the non-mevalonate pathway of  Hevea brasiliensis  was obtained and nucleotide sequences of these genes were determined. The gene cluster according to this invention involved in the IPP biosynthesis in the non-mevalonate pathway is involved in the biosynthesis of vitamin E and carotenoids. Therefore, the  Hevea brasiliensis  obtained by introducing the gene cluster of the present invention can be expected to produce high-quality rubber with improved permanence.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a gene cluster involved in biosynthesisof isopentenyl diphosphate in the non-mevalonate pathway of Heveabrasiliensis.

2. Background Art

All kinds of steroids, terpenoids, carotinoids, and all kinds ofvitamins are composed of plural 5-carbon isoprenes covalently bound toone another. The basic isoprene structure is called “isoprene unit”, anda compound having isoprene unit is generically called “isoprenoid”.Isopentenyl diphosphate (IPP), a compound having five-carbons, serves asa unit in condensation reaction when an isoprenoid compound issynthesized. The two pathways of mevalonate pathway and thenon-mevalonate pathway are known as the IPP biosynthesis pathways. Inplants, it is said that the mevalonate pathway functions in cellcytoplasm and the non-mevalonate pathway functions in plastids. InEscherichia coli, each of the non-mevalonate pathway genes is isolated,and its functions are confirmed. In Hevea brasiliensis, on the otherhand, the sequences of the gene cluster have not been reported. FIG. 1shows the non-mevalonate pathway of IPP synthesis. Details aboutisoprenoid biosynthesis by the non-mevalonate pathway are given in thegeneral remarks in W. Eisenreich et al., Cell Mol. Life Sci. 61 (2004)1401-1426, for example.

In the non-mevalonate pathway, firstly glyceraldehyde 3-phosphate andpyruvic acid are catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase(DXS) to undergo condensation accompanied by decarboxylation reaction,thereby 1-deoxy-D-xylulose 5-phosphate (DXP) is formed. DXS catalyzestransfer of 2 carbonates using thiamine diphosphate as a cofactor by thereaction mechanism similar to transketolase and pyruvic aciddecarboxylase.

DXP undergoes transferring reaction and then reduced through catalysisby 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), thereby forms2-C-methyl-D-erythritol-4-phosphate (MEP). MEP is conjugated with CDP byMEP cytidyltransferase (MCT) to form4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol (MEPPC). The3-Hydroxyl group of MEPPC is phosphorylated by4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK), andMEPPC is converted to 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol2-phosphate (MEPPCP).

MEPPCP is catalyzed by 2-C-methylerythritol 2,4-cyclodiphosphatesynthase (MECPS) to form 2-C-methylerythritol 2,4-cyclodiphosphate(MECPP). Next, MSCPP is reductively converted to1-hydroxy-2-methyl-2-(E)-butenyl 4-phosphate (HMBPP). Further, IPP andDMAPP are biosynthesized from HMBPP.

SUMMARY OF THE INVENTION

Now, it is an object of the present invention to isolate the genecluster involved in the biosynthesis of IPP in the non-mevalonatepathway of Hevea brasiliensis, and to analyze the nucleotide sequence ofeach of the genes composing the gene cluster.

The sequences which are assumed to be the gene cluster involved in thebiosynthesis of IPP in the non-mevalonate pathway are identified bysyntactic analysis of the information on the gene fragment obtainedthrough EST (Expression Sequence Tags) analysis of Hevea brasiliensisand known gene databases, and the gene homologs in relation to thenon-mevalonate pathway are obtained by full-length cDNA cloning. Thenthe nucleotide sequences of each of the obtained genes are determined.

The gene cluster of the present invention involved in the biosynthesisof IPP by the non-mevalonate pathway is involved in the biosynthesis ofvitamin E and carotenoids, so useful plants containing vitamin E andcarotenoids at a high amount can be produced by transforming plants bythe gene cluster obtained by the present invention. More specifically,the transformed Hevea brasiliensis obtained by introducing the genes ofthe present invention can be expected to produce a high-quality rubberwith improved permanence. Especially, accompanied with the increasedvitamin E content, many effects can be expected, including reduction ofthe quantity of synthetic antioxidants added when processing rubber.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a figure showing the details of the mevalonate pathway.

FIG. 2 is a photograph showing the result of complementation assay of1-deoxy-D-xylulose-5-phosphate reductoisomerase.

FIG. 3 is a photograph showing the result of complementation assay of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase.

FIG. 4 is a photograph showing the result of complementation assay of2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to achieve the object above, the inventors of the presentinvention have determined gene nucleotide sequences by EST analysis andcDNA cloning. Total RNA was extracted from latex of the standard treeand xylem of the current year branch of Hevea brasiliensis so as toprepare cDNA libraries. Exhaustive one-pass sequence analysis wasperformed on these libraries. Then 16407 EST sequences were obtainedfrom the cDNA library prepared from the latex and 16305 EST sequencesfrom the cDNA library were obtained from the xylem with high accuracy(Total 32442). On the obtained partial sequences, clustering analysisbased on similarity between sequences and annotation analysis based oncomparison with known genes were performed, and thus an EST database ofHevea brasiliensis was constructed.

In the obtained EST database, the inventors have found EST sequenceswhich are thought to encode enzymes of the non-mevalonate pathway, morespecifically, 1-deoxy-D-xylulose-5-phosphate synthase,1-deoxy-D-xylulose-5-phosphate reductoisomerase,2-C-methyl-D-erythritol-4-phosphate cytidyltransferase,4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase,2-C-methyl-D-erythritol 2,4-cyclodiphosphate syntase,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, and1-hydroxy-2-methyl-butenyl-4-diphosphate reductase. Moreover, as tothese sequences, the inventors have determined the 3′terminal sequenceby 3′-RACE (Rapid Amplification of cDNA Ends) and obtained full-lengthcDNAs.

A gene encoding 1-deoxy-D-xylulose-5-phosphate synthase is representedby nucleotide numbers from 1 to 2591 in SEQ ID NO:1 in the sequencelist. The part corresponding to nucleotide numbers from 235 to 2394 inthe nucleotide sequence in SEQ ID NO:1 in the sequence list correspondsto the open reading frame. A deduced amino acid sequence of1-deoxy-D-xylulose-5-phosphate synthase obtained from the nucleotidesequence of the open reading frame is represented by amino acid numbersfrom 1 to 720 in SEQ ID NO:2 in the sequence list. Meanwhile, the1-deoxy-D-xylulose-5-phosphate synthase is an enzyme that catalyzes thereaction which biosynthesizes 1-deoxy-D-xylulose-5-phosphate usingpyruvic acid and glyceraldehyde-3-phosphate as substrate.

A gene encoding 1-deoxy-D-xylulose-5-phosphate reductoisomerase isrepresented by nucleotide numbers from 1 to 1929 in SEQ ID NO:3 in thesequence list. Meanwhile, this sequence contains the sequence of avector, and the part excluded with the vector part corresponds tonucleotide numbers from 1 to 1884 in the nucleotide sequence in SEQ IDNO:3 in the sequence list. The part corresponding to nucleotide numbersfrom 301 to 1713 in the nucleotide sequence in SEQ ID NO:3 in thesequence list corresponds to an open reading frame. Meanwhile, this partcontains the sequence of a vector, and the open reading frame excludedwith the vector corresponds to nucleotide numbers from 256 to 1671 inthe nucleotide sequence in SEQ ID NO:3 in the sequence list. A deducedamino acid sequence of 1-deoxy-D-xylulose-5-phosphate reductoisomeraseobtained from the nucleotide sequence of the open reading frame isrepresented by amino acid numbers from 1 to 471 in SEQ ID NO:4 in thesequence list. Meanwhile, the 1-deoxy-D-xylulose-5-phosphatereductoisomerase is an enzyme that catalyzes the reaction whichbiosynthesizes 2-C-methyl-D-erythritol-4-phosphate using1-deoxy-D-xylulose-5-phosphate as substrate.

A Gene encoding 2-C-methyl-D-erythritol-4-phosphate cytidyltransferaseis represented by nucleotide numbers from 1 to 1335 in SEQ ID NO:5 inthe sequence list. Meanwhile, this sequence contains the sequence of avector, and the part excluded with the vector part corresponds tonucleotide numbers from 1 to 1301 in the nucleotide sequence in SEQ IDNO:5 in the sequence list. The part corresponding to nucleotide numbersfrom 214 to 1146 in the nucleotide sequence in SEQ ID NO:5 in thesequence list corresponds to an open reading frame. Meanwhile, this partcontains the sequence of a vector, and the open reading frame excludedwith the vector corresponds to nucleotide numbers from 180 to 1115 inthe nucleotide sequence in SEQ ID NO:5 in the sequence list. A deducedamino acid sequence of 2-C-methyl-D-erythritol-4-phosphatecytidyltransferase obtained from the nucleotide sequence of the openreading frame is represented by amino acid numbers from 1 to 311 in SEQID NO:6 in the sequence list. Meanwhile, the2-C-methyl-D-erythritol-4-phosphate cytidyltransferase is an enzyme thatcatalyzes the reaction which biosynthesizes4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol using2-C-methyl-D-erythritol-4-phosphate as substrate.

A Gene encoding 2-C-methyl-D-erythritol-4-phosphate cytidyltransferaseobtained from another clone is represented by nucleotide numbers from 1to 2069 in SEQ ID NO:7 in the sequence list. Meanwhile, this sequencecontains the sequence of a vector, and the part excluded with the vectorpart corresponds to nucleotide numbers from 1 to 1254 in the nucleotidesequence in SEQ ID NO:7 in the sequence list. The part corresponding tonucleotide numbers from 185 to 1117 in the nucleotide sequence in SEQ IDNO:7 in the sequence list corresponds to an open reading frame.Meanwhile, this part contains the sequence of a vector, and the openreading frame excluded with the vector corresponds to nucleotide numbersfrom 150 to 1085 in the nucleotide sequence in SEQ ID NO:7 in thesequence list. A deduced amino acid sequence of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase obtained from thenucleotide sequence of the open reading frame is represented by aminoacid numbers from 1 to 311 in SEQ ID NO:8 in the sequence list.Meanwhile, the 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase isan enzyme that catalyzes the reaction which biosynthesizes4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol using2-C-methyl-D-erythritol-4-phosphate as substrate.

A gene encoding 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinaseis represented by nucleotide numbers from 1 to 1512 in SEQ ID NO:9 inthe sequence list. The part corresponding to nucleotide numbers from 110to 1276 in the nucleotide sequence in SEQ ID NO:9 in the sequence listcorresponds to the open reading frame. A deduced amino acid sequence of4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase obtained fromthe nucleotide sequence of the open reading frame is represented byamino acid numbers from 1 to 388 in SEQ ID NO:10 in the sequence list.Meanwhile, the 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinaseis an enzyme that catalyzes the reaction which biosynthesizes4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol diphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol as substrate.

A gene encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase isrepresented by nucleotide numbers from 1 to 1036 in SEQ ID NO:11 in thesequence list. The part corresponding to nucleotide numbers from 1 to714 in the nucleotide sequence in SEQ ID NO:11 in the sequence listcorresponds to the open reading frame. A deduced amino acid sequence of2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase obtained from thenucleotide sequence of the open reading frame is represented by aminoacid numbers from 1 to 237 in SEQ ID NO:12 in the sequence list.Meanwhile, the 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase isan enzyme that catalyzes the reaction which biosynthesizes2-C-methyl-D-erythritol 2,4-cyclodiphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythrithol diphosphate assubstrate.

A gene encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthaseobtained from another clone is represented by nucleotide numbers from 1to 989 in SEQ ID NO:13 in the sequence list. The part corresponding tonucleotide numbers from 49 to 774 in the nucleotide sequence in SEQ IDNO:13 in the sequence list corresponds to the open reading frame. Adeduced amino acid sequence of 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase obtained from the nucleotide sequence ofthe open reading frame is represented by amino acid numbers from 1 to241 in SEQ ID NO:14 in the sequence list. Meanwhile, the2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase is an enzyme thatcatalyzes the reaction which biosynthesizes 2-C-methyl-D-erythritol2,4-cyclodiphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythrithol diphosphate assubstrate.

A gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthaseis represented by nucleotide numbers from 1 to 2745 in SEQ ID NO:15 inthe sequence list. Meanwhile, this sequence contains the sequence of avector, and the part excluded with the vector part corresponds tonucleotide numbers from 1 to 2713 in the nucleotide sequence in SEQ IDNO:15 in the sequence list. The part corresponding to nucleotide numbersfrom 184 to 2403 in the nucleotide sequence in SEQ ID NO:15 in thesequence list corresponds to an open reading frame. Meanwhile, this partcontains the sequence of a vector, and the open reading frame excludedwith the vector corresponds to nucleotide numbers from 152 to 2374 inthe nucleotide sequence in SEQ ID NO:15 in the sequence list. A deducedamino acid sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphatesynthase obtained from the nucleotide sequence of the open reading frameis represented by amino acid numbers from 1 to 740 in SEQ ID NO:16 inthe sequence list. Meanwhile, the1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase is an enzymethat catalyzes the reaction which biosynthesizes1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate using2-C-methyl-D-erythritol-2,4-cyclodiphosphate as substrate.

A gene encoding 1-hydroxy-2-methyl-butenyl-4-diphosphate reductase isrepresented by nucleotide numbers from 1 to 1682 in SEQ ID NO:17 in thesequence list. Meanwhile, this sequence contains the sequence of avector, and the part excluded with the vector part corresponds tonucleotide numbers from 1 to 1632 in the nucleotide sequence in SEQ IDNO:17 in the sequence list. The part corresponding to nucleotide numbersfrom 107 to 1492 in the nucleotide sequence in SEQ ID NO:17 in thesequence list corresponds to an open reading frame. Meanwhile, this partcontains the sequence of a vector, and the open reading frame excludedwith the vector corresponds to nucleotide numbers from 57 to 1445 in thenucleotide sequence in SEQ ID NO:17 in the sequence list. A deducedamino acid sequence of 1-hydroxy-2-methyl-butenyl-4-diphosphatereductase obtained from the nucleotide sequence of the open readingframe is represented by amino acid numbers from 1 to 462 in SEQ ID NO:18in the sequence list. Meanwhile, the1-hydroxy-2-methyl-butenyl-4-diphosphate reductase is an enzyme thatcatalyzes the reaction which biosynthesizes isopentenyl diphosphate anddimethylallyl diphosphate using1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate as substrate.

According to recombinant DNA techniques, artificial mutation can be madeto a particular site of the original DNA, without changing thefundamental properties of the DNA or in such a way as to improve theseproperties. As to genes having natural nucleotide sequences providedaccording to the present invention or even genes having nucleotidesequences different from the natural sequence, artificial insertion,deletion and substitution can be performed in the same manner, and theycan be altered to have an equal or improved properties as the naturalgenes. Moreover, the present invention includes such mutated genes.

More specifically, a gene consisting of a nucleotide sequence in which apart of the nucleotide sequence shown in SEQ ID NO:1 in the sequencelist has been deleted, substituted or added means a gene in which nomore than 20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:1 have beensubstituted. Furthermore, such a gene has no less than 95%, preferablyno less than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:1. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 1-deoxy-D-xylulose-5-phosphate synthase,which biosynthesizes 1-deoxy-D-xylulose-5-phosphate using pyruvic acidand glyceraldehyde-3-phosphate as substrate. Additionally, such a genehybridizes with the gene shown in SEQ ID NO:1 under stringentconditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:3 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:3 have beensubstituted. Furthermore, such a gene has no less than 95%, preferablyno less than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:3. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 1-deoxy-D-xylulose-5-phosphatereductoisomerase, which biosynthesizes2-C-methyl-D-erythritol-4-phosphate using 1-deoxy-D-xylulose-5-phosphateas substrate. Additionally, such a gene hybridizes with the gene shownin SEQ ID NO:3 under stringent conditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:5 in the sequence list hasbeen deleted, substituted or means a gene in which no more than 20,preferably no more than 10, more preferably no more than 5 nucleotidesequences in the nucleotide sequence in SEQ ID NO:5 have beensubstituted. Furthermore, such a gene has no less than 95%, preferablyno less than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:5. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 2-C-methyl-D-erythritol-4-phosphatecytidyltransferase, which biosynthesizes4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol using2-C-methyl-D-erythritol-4-phosphate as substrate. Additionally, such agene hybridizes with the gene shown in SEQ ID NO:5 under stringentconditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:7 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:7 have beensubstituted. Further, such a gene has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:7. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 2-C-methyl-D-erythritol-4-phosphatecytidyltransferase, which biosynthesizes4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol using2-C-methyl-D-erythritol-4-phosphate as substrate. Additionally, such agene hybridizes with the gene shown in SEQ ID NO:7 under stringentconditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:9 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:9 have beensubstituted. Further, such a gene has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:9. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase, whichbiosynthesizes4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol-diphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol as substrate.Additionally, such a gene hybridizes with the gene shown in SEQ ID NO:9under stringent conditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:11 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:11 havebeen substituted. Further, such a gene has no less than 95%, preferablyno less than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:11. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase, which biosynthesizes2-C-methyl-D-erythritol 2,4-cyclodiphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythrithol diphosphate assubstrate. Additionally, such a gene hybridizes with the gene shown inSEQ ID NO:11 under stringent conditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:13 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:13 havebeen substituted. Further, such a gene has no less than 95%, preferablyno less than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:13. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase, which biosynthesizes2-C-methyl-D-erythritol 2,4-cyclodiphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythrithol diphosphate assubstrate. Additionally, such a gene hybridizes with the gene shown inSEQ ID NO:13 under stringent conditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:15 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:15 aresubstituted. Further, such a gene has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:15. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase whichbiosynthesizes 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate using2-C-methyl-D-erythritol-2,4-cyclodiphosphate as substrate. Additionally,such a gene hybridizes with the gene shown in SEQ ID NO:15 understringent conditions.

Similarly, a gene consisting of a nucleotide sequence in which a part ofthe nucleotide sequence shown in SEQ ID NO:17 in the sequence list hasbeen deleted, substituted or added means a gene in which no more than20, preferably no more than 10, more preferably no more than 5nucleotide sequences in the nucleotide sequence in SEQ ID NO:17 havebeen substituted. Further, such a gene has no less than 95%, preferablyno less than 97%, more preferably no less than 99% homology with thenucleotide sequence shown in SEQ ID NO:17. Even such a gene is alsowithin the scope of the present invention, as long as the gene encodes aprotein having the function as 1-hydroxy-2-methyl-butenyl-4-diphosphatereductase which biosynthesizes isopentenyl diphosphate and dimethylallyldiphosphate using 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate assubstrate. Additionally, such a gene hybridizes with the gene shown inSEQ ID NO:17 under stringent conditions.

Those skilled in the art may select conditions for hybridization adlibitum. A membrane onto which a DNA or RNA molecule to be tested hasbeen transferred and a labeled probe can be hybridized in an applicablehybridization buffer. The hybridization buffer may be composed of 5×SSC,0.1 weight % N-lauroyl sarcosine, 0.02 weight % SDS, 2 weight % blockingreagent for nucleotide sequence hybridization, and 50 weight %formamide, for instance. As the blocking reagent for nucleotide sequencehybridization, for example, commercially available blocking reagent fornucleotide sequence hybridization can be dissolved into a buffersolution (pH 7.5) composed of 0.1 M maleic acid and 0.15 M NaCl to makethe concentration of the blocking reagent to be 10%. 20×SSC may becomposed of 3M NaCl and 0.3M citric acid solution. SSC may be usedpreferably at 3 to 6×SSC concentration, and more preferably at 4 to5×SSC concentration.

Hybridization may be performed at 40 to 80° C., preferably at 50 to 70°C., and more preferably at 55 o 65° C. Washing may be performed using awashing buffer after incubation for several hours or overnight. Washingmay be performed preferably at room temperature, and more preferably atthe temperature of hybridization. The washing buffer may be composed of6×SSC+0.1 weight % SDS solution, preferably composed of 4×SSC+0.1 weight% SDS solution, more preferably composed of 2×SSC+0.1 weight % SDSsolution, even more preferably composed of 1×SSC+0.1 weight % SDSsolution, and most preferably composed of 0.1×SSC+0.1 weight % SDSsolution. The membrane can be washed with such a washing buffer and theDNA molecule or RNA molecule hybridized with the probe can be identifiedby the label used for the probe.

Further herein, a protein consisting of an amino acid sequence in whicha part of the amino acid sequence shown in SEQ ID NO:2 has been deleted,substituted or added means a protein in which no more than 20,preferably no more than 10, more preferably no more than 5 amino acidsin the amino acid sequence in SEQ ID NO:2 have been substituted.Further, such a protein has no less than 95%, preferably no less than97%, more preferably no less than 99% homology with the amino acidsequence shown in SEQ ID NO:2. Even such a protein is within the scopeof the present invention, as long as the protein has the function as1-deoxy-D-xylulose-5-phosphate synthase, which biosynthesizes1-deoxy-D-xylulose-5-phosphate using pyruvic acid andglyceraldehyde-3-phosphate as substrate.

Further herein, a protein consisting of an amino acid sequence in whicha part of the amino acid sequence shown in SEQ ID NO:4 has been deleted,substituted or added means a protein in which no more than 20,preferably no more than 10, more preferably no more than 5 amino acidsin the amino acid sequence in SEQ ID NO:4 have been substituted.Further, such a protein has no less than 95%, preferably no less than97%, more preferably no less than 99% homology with the amino acidsequence shown in SEQ ID NO:4. Even such a protein is within the scopeof the present invention, as long as the protein has the function as1-deoxy-D-xylulose-5-phosphate reductoisomerase which biosynthesizes2-C-methyl-D-erythritol-4-phosphate using 1-deoxy-D-xylulose-5-phosphateas substrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:6 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:6 have beensubstituted. Further, such a protein has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with the aminoacid sequence shown in SEQ ID NO:6. Even such a protein is within thescope of the present invention, as long as the protein has the functionas 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase whichbiosynthesizes 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol using2-C-methyl-D-erythritol-4-phosphate as substrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:8 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:8 have beensubstituted. Further, such a protein has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with the aminoacid sequence shown in SEQ ID NO:8. Even such a protein is within thescope of the present invention, as long as the protein has the functionas 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase whichbiosynthesizes 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol using2-C-methyl-D-erythritol-4-phosphate as substrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:10 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:10 have beensubstituted. Further, such a protein has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with the aminoacid sequence shown in SEQ ID NO:10. Even such a protein is within thescope of the present invention, as long as the protein has the functionas 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase whichbiosynthesizes 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritoldiphosphate using 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol assubstrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:12 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:12 have beensubstituted. Further, such a protein has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with the aminoacid sequence shown in SEQ ID NO:12. Even such a protein is within thescope of the present invention, as long as the protein has the functionas 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase whichbiosynthesizes 2-C-methyl-D-erythritol 2,4-cyclodiphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythrithol diphosphate assubstrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:14 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:14 have beensubstituted. Further, such a protein has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with the aminoacid sequence shown in SEQ ID NO:14. Even such a protein is within thescope of the present invention, as long as the protein has the functionas 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase whichbiosynthesizes 2-C-methyl-D-erythritol 2,4-cyclodiphosphate using4-(cytidine-5′-diphospho)-2-C-methyl-D-erythrithol diphosphate assubstrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:16 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:16 have beensubstituted. Further, such a protein has have no less than 95%,preferably no less than 97%, more preferably no less than 99% homologywith the amino acid sequence shown in SEQ ID NO:16. Even such a proteinis within the scope of the present invention, as long as the protein hasthe function as 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthasewhich biosynthesizes 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphateusing 2-C-methyl-D-erythritol-2,4-cyclodiphosphate as substrate.

Similarly, a protein consisting of an amino acid sequence in which apart of the amino acid sequence shown in SEQ ID NO:18 in the sequencelist has been deleted, substituted or added means a protein in which nomore than 20, preferably no more than 10, more preferably no more than 5amino acids in the amino acid sequence in SEQ ID NO:18 have beensubstituted. Further, such a protein has no less than 95%, preferably noless than 97%, more preferably no less than 99% homology with the aminoacid sequence shown in SEQ ID NO:18. Even such a protein is within thescope of the present invention, as long as the protein has the functionas 1-hydroxy-2-methyl-butenyl-4-diphosphate reductase whichbiosynthesizes isopentenyl diphosphate and dimethylallyl diphosphateusing 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate as substrate.

The gene cluster of the present invention involved in the biosynthesisof isopentenyl diphosphate in the non-mevalonate pathway can beintroduced into plants such as rubber tree to enhance its expression, sothat gene products from the non-mevalonate pathway can be increased insaid plants. Not polyisoprene (rubber component), which is the majorcomponent of latex, but many non-rubber components can be synthesized inthe non-mevalonate pathway. Especially, tocotrienol and carotenoid,which are vitamin Es, are known to exert antioxidant effects in naturalrubber made from processed latex, so if this component is increased,improvement in permanence of rubber material can be expected. Furtherherein, “to improve the property of the rubber” means to have desiredeffects of improving permanence on the rubber material obtained byincreasing Vitamin E or carotenoid content in natural rubber.

The plant to be introduced with gene of the present invention is notlimited to Hevea brasiliensis, the examples of other plants may beguayule, cassava, sunflower, lettuce, Indian rubber tree, and etc., butthe target plants to be transformed are not limited to these plants, andtransformants into which the gene of the present invention has beenintroduced can be produced in various plants. Particularly, according tothis invention, it is preferable to transform rubber-producing plants,such as Hevea Brasiliensis, in order to improve the quality of therubber obtained from said rubber-producing plants. Rubber-producingplants are known to spread wide variety of families includingAsteraceae, Moraceae, Euphorbiaceae, Asclepiadaceae, and Apocynaceae.

As a method for producing transformants, usual methods known in the artcan be used. As an useful promoter for activating the introduced gene,the cauliflower mosaic virus 35S promoter widely used in the art, forexample, can be used and positioned upstream of the gene of the presentinvention, which is to be transduced. In many cases, some promoter isrequired to achieve sufficient expression of the introduced foreigngene. The preferred promoter is not limited to the cauliflower mosaicvirus 35S promoter, and various promoters widely used in the art mayalso be used.

Furthermore, the vectors which can be used in the present invention mayinclude, but not limited to, vectors such as pIG121-Hm, pBI12, pBI221,pBIN19, pCC22, pGA482, pPCV001, pCGN1547, pJJ1881, pPZP111, pGreen0029,pBI101, pBI121, and pYLTAC7. Transgenic plants can be prepared byintroducing such vectors into Agrobacterium, for instance, to have acallus or a plantlet infected, and thus seeds derived from suchtransgenic plants can be obtained. Further, the transformation methodfor introducing the plant gene of the present invention into plants isnot limited to the Agrobacterium method, but various methods commonlyused in the art including the particle gun method and theelectroporation method may also be used. Additionally, an example inwhich a foreign gene is introduced into rubber tree for transformationis disclosed in Japanese Patent Publication No. 1996-116977. Therefore,those skilled in the art can produce a transgenic plant, into which thegene of the present invention has been introduced, by making appropriatealterations with reference to the description of Japanese PatentPublication No. 1996-116977.

EXAMPLES

The present invention will be specifically described below withreference to examples, but the scope of the present inventions will notbe limited to these examples.

(Materials)

Latex and xylem from the current year branch of Hevea brasiliensisstandard tree PRIM 600 cultivated in Cikampek, Indonesia were used as aplant sample. The latex was suspended in an equal amount of 2× samplingbuffer (0.1M Tris-HCL, 0.3M LiCl, 0.01M EDTA, 10% SDS) immediately aftersampling the latex. Also, a mutant E. coli strain used was gifted fromassociate professor Tomohisa Kuzuyama, Biotechnology Research Center ofthe University of Tokyo.

(RNA Extraction from Hevea brasiliensis)

RNA was extracted from the latex and xylem respectively by the followingprocedures. Immediately after sampling, the sample (equivalent to 25 mlof latex) suspended in an equal amount of 2× sampling buffer (0.1MTris-HCL, 0.3M LiCl, 0.01M EDTA, 10% SDS) was centrifuged, and the upperlayer constituting the rubber layer was removed. Then, 1.5 equivalentamount of 2×CTAB solution (2% Hexadecyltrimethylammonium bromide (CTAB),1% 2-mercaptoethanol, 0.1 M Tris-HCL (pH9.5), 1.4 M NaCl, 20 mM EDTA)was added. After incubating at 65° C. for 10 minutes, treatment withchloroform/isoamyl alcohol was performed (repeated twice). A ¼ amount of10 M LiCl was added to the collected aqueous layer and mixed, thenincubated at −20° C. for 2 hours (selective precipitation of RNA). Itwas centrifuged, the precipitation was dissolved into an appropriateamount of TE, then centrifuged, and the supernatant was collected(polysaccharides were removed). Further, the fraction was treated withphenol, phenol/chloroform, chloroform/isoamyl alcohol, and thenselective precipitation of RNA by LiCl was performed again. Theprecipitation was cleaned with 70% ethanol, and dissolved inDEPC-treated water after being dried under reduced pressure. Thus, totalRNA derived from latex was obtained.

Also, the phloem of the current year branch was peeled off by a knife toobtain about 1 g of xylem, and it was pound in a mortar with a pestlewhile cooling with liquid nitrogen. The total RNA derived from xylem wasobtained using RNeasy Plant Mini Kit (registered trademark, Qiagen).

The obtained RNA solution was quantified by optical densitymeasurements, and this was confirmed by electrophoresis. A 450 μg of RNAwas obtained from 25 mg of latex, and 110 μg of RNA was obtained from 1g of xylem.

(Preparation of cDNA Libraries of Hevea brasiliensis)

The cDNA libraries were prepared from the RNA samples derived from Heveabrasiliensis latex and xylem by the G-Capping method at HitachiInstruments Service Co., Ltd. The G-Capping method is a method that canachieve full-length cDNAs at a high percentage.

The cDNA library derived from the latex has the library size of 1.7×10⁵,the insert percentage of 71% (24 samples/agarose gel electrophoresis),and the percentage of full-length cDNA was 82% (toward clones withinsert). The size of cDNA library derived from the xylem was 2.9×10⁵,and the percentage of insert was 80% (24 samples/agarose gelelectrophoresis), and the percentage of full-length cDNA was 87% (towardclones with insert).

(Sequence Analysis, Clustering Analysis and Annotation Analysis of ESTSequences)

At the Genome Information Science Laboratory of Kitasato Institute forLife Sciences of Kitasato University, exhaustive one-pass sequenceanalysis was performed on approximately 20,000 clones of the cDNAlibraries derived from latex and xylem of Hevea brasiliensisrespectively. According to the sequence information obtained from thesequence analysis, clones with no insert and clones failed to determinesequence were removed, then high accuracy sequence information wasobtained. The latex cDNA library and xylem cDNA library provided 16407EST sequences and 16305 EST sequences respectively with high accuracy(total 32442).

The obtained partial sequences were subjected to clustering analysisbased on similarity between sequences, and annotation analysis based oncomparison with known genes, thereby an EST database of Heveabrasiliensis was constructed. A VISUALBIO clustering of NTT Software wasused for the clustering analysis. The annotation analysis was performedby homology search using NCBI BLAST. The database used for the searchwas nr (All non-redundant GenBank CDS translations+PDB+SwissProt+PIR(Peptide Sequence Database)).

In the obtained EST database, EST sequences of the enzymes involved inthe non-mevalonate pathway were found, i.e.1-deoxy-D-xylulose-5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphatereductoisomerase, 2-C-methyl-D-erythritol-4-phosphatecytidylyltransferase, 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritolkinase, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase,1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase, and1-hydroxy-2-methyl-butenyl-4-diphosphate reductase.

(Determination of Sequences at the 3′ Terminal by 3′-RACE)

The sequences at the 3′ terminal were determined by 3′-RACE (RapidAmplification of cDNA Ends) on each sequences obtained by the analysesabove to obtain full-length cDNAs. For 3′-RACE, a 3′-Full RACE Core Set(Takara Bio Inc.) was used. An oligo-dT primer was used for reversetranscription. For amplification by PCR, an oligo-dT primer and a senseprimer having sequence identity with a part of known sequences wereused. The amplified fragments were obtained from reverse transcriptionand PCR, then the fragments were subjected to TA cloning into pT7Bluevector, which was succeeded by sequence analysis.

The sequences obtained in this way are as follows; (1) the nucleotidesequence of 1-deoxy-D-xylulose-5-phosphate synthase gene (SEQ ID NO:1 inthe sequence list), (2) the nucleotide sequence of1-deoxy-D-xylulose-5-phosphate reductoisomerase gene (SEQ ID NO:3 in thesequence list), (3) the nucleotide sequence of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase gene (SEQ ID NO:5in the sequence list), (4) the nucleotide sequence of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase gene derived fromanother clone (SEQ ID NO:7 in the sequence list), (5) the nucleotidesequence of 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinasegene (SEQ ID NO:9 in the sequence list), (6) the nucleotide sequence of2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene (SEQ ID NO:11in the sequence list), (7) the nucleotide sequence of2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase gene derived fromanother clone (SEQ ID NO:13 in the sequence list), (8) the nucleotidesequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene(SEQ ID NO:15 in the sequence list), and (9) the nucleotide sequence of1-hydroxy-2-methyl-butenyl-4-diphosphate reductase gene (SEQ ID NO:17 inthe sequence list).

Additionally, the deduced amino acid sequences of the proteins obtainedfrom the open reading frames of these nucleotide sequences are asfollows; (1) the amino acid sequence of 1-deoxy-D-xylulose-5-phosphatesynthase (SEQ ID NO:2 in the sequence list), (2) the amino acid sequenceof 1-deoxy-D-xylulose-5-phosphate reductoisomerase (SEQ ID NO:4 in thesequence list), (3) the amino acid sequence of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (SEQ ID NO:6 inthe sequence list), (4) the amino acid sequence of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase derived fromanother clone (SEQ ID NO:8 in the sequence list), (5) the amino acidsequence of 4-(cytidine-5′-diphospho)-2-C-methyl-D-erythritol kinase(SEQ ID NO:10 in the sequence list), (6) the amino acid sequence of2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (SEQ ID NO:12 inthe sequence list), (7) the amino acid sequence of2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase derived fromanother clone (SEQ ID NO:14 in the sequence list), (8) the amino acidsequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (SEQID NO:16 in the sequence list), and (9) the amino acid sequence of1-hydroxy-2-methyl-butenyl-4-diphosphate reductase (SEQ ID NO:18 in thesequence list).

(Complementary Assay Using Transformed E. coli)

Among sequences obtained by the procedure as described above, thefunctions of the genes encoding 1-deoxy-D-xylulose-5-phosphatereductoisomerase, 2-C-methyl-D-erythritol-4-phosphate cytidyltransferaseand 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase were confirmedby complementary assay using transformed E. coli strains deficient ofthe above-mentioned corresponding genes.

PCR was performed using sense primers and antisense primers attachedwith appropriate restriction sites, the sequences corresponding to thereading frames of 1-deoxy-D-xylulose-5-phosphate reductoisomerase,2-C-methyl-D-erythritol-4-phosphate cytidyltransferase and2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase were amplifiedrespectively. The amplified fragments were in frame cloned into pMW118vector (Nippon gene).

The genes subjected to the analysis are indispensable for growth of E.coli strain. The strain deficient of 1-deoxy-D-xylulose-5-phosphatereductoisomerase gene, which was used as the background of thecomplementary assay, can grow on culture medium by adding2-C-methylerythritol into the culture medium. Moreover, the strainsdeficient of 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase andthe strain deficient of 2-C-methyl-D-erythritol 2,4-cyclodiphosphatesynthase are introduced with a plasmid (pTMV20KM) containing the genecluster involved in mevalonate pathway derived from Streptomyces sp.CL190 strain. Therefore, the strains can grow by adding mevalonic acidinto the medium owing to the enzymes involved in the mevalonic acidintroduced into the strains. These E. coli mutant strains were giftedfrom associate professor Tomohisa Kuzuyama, Biotechnology ResearchCenter of the University of Tokyo. Meanwhile, the strain deficient of1-deoxy-D-xylulose-5-phosphate reductoisomerase used in this study isdescribed in Kuzuyama et al., Biosci. Biotechnol. Biochem., 63(4),776-778.1999, and the strains deficient of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase and the straindeficient of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase aredescribed in Takagi et al., Tetrahedron Letters 41(2000)3395-3398,respectively.

The pMW118 vector was introduced into mutant strains, and the vectorcontained the reading frame region of the target gene derived from HeveaBrasiliensis. If the function of the transformed E. coli strain iscomplemented by the introduced target gene from Hevea Brasiliensis, itis assumed that the transformed E. coli may recover the ability to growon the normal LB medium not containing 2-C-methylerythritol or mevalonicacid. By performing the complementation assay to identify the functionsof the target genes, the functions of 1-deoxy-D-xylulose-5-phosphatereductoisomerase (one clone), 2-C-methyl-D-erythritol-4-phosphatecytidyltransferase (two clones) and 2-C-methyl-D-erythritol2,4-cyclodiphosphate synthase (two clones) were confirmed. The concretedata will be described below.

The data on the complementary assay of 1-deoxy-D-xylulose-5-phosphatereductoisomerase is shown in FIG. 2, that of2-C-methyl-D-erythritol-4-phosphate cytidyltransferase is shown in FIG.3, and that of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase isshown in FIG. 4, respectively.

As well, in FIG. 2, the data corresponding to the portion 1 of the petridish shows the series of the wild-type strain, the data corresponding tothe portion 2 of the petri dish shows the series of the transformed E.coli strain, the data corresponding to the portion 3 of the petri dishshows the series of the transformed E. coli strain added with a genomicfragment of E. coli gene, the data corresponding to the portion 4 of thepetri dish shows the series of the transformed E. coli strain added withan open reading frame from E. coli gene, the data corresponding to theportion 5 of the petri dish shows the series of the transformed E. colistrain added with an open reading frame from Arabidopsis gene, the datacorresponding to the portion 6 of the petri dish shows the series of thetransformed E. coli strain added with an open reading frame fromArabidopsis gene deleted with the signal sequence, the datacorresponding to the portion 7 of the petri dish shows the series of thetransformed E. coli strain added with an open reading frame from Heveabrasiliensis gene, and the data corresponding to the portion 8 of thepetri dish shows the series of the transformed E. coli strain added withthe open reading frame of Arabidopsis gene deleted with the signalsequence, respectively.

Moreover, in FIGS. 3 and 4, the data corresponding to the portion 1 ofthe petri dish shows the series of the wild-type strain, the datacorresponding to the portion 2 of the petri dish shows the series of thetransformed E. coli strain, the data corresponding to the portion 3 ofthe petri dish shows the series of the transformed E. coli strain addedwith a genomic fragment of E. coli gene, the data corresponding to theportion 4 of the petri dish shows the series of the transformed E. colistrain added with an open reading frame from E. coli gene, the datacorresponding to the portion 5 of the petri dish shows the series of thetransformed E. coli strain added with an open reading frame fromArabidopsis gene, the data corresponding to the portion 6 of the petridish shows the series of the transformed E. coli strain added with anopen reading frame from Arabidopsis gene deleted with the signalsequence, the data corresponding to the portions 7 and 9 of the petridish show the series of the transformed E. coli strain added with anopen reading frame from Hevea brasiliensis gene, and the datacorresponding to the portions 8 and 10 of the petri dish show the seriesof the transformed E. coli strain added with an open reading frame ofArabidopsis gene deleted with the signal sequence, respectively.

As shown from FIG. 2 to FIG. 4, the wild-type strain grew normally asshown in portion 1 of the petri dish, while the E. coli mutant straincould not grow normally in this medium as shown in portion 2 of thepetri dish. However, the E. coli mutant strain recovered the ability ofgrowing by compensating the gene to be tested, indicatingcomplementation of the deficient gene.

According to the present invention, the gene cluster involved in thenon-mevalonate pathway of Hevea brasiliensis was obtained, and thenucleotide sequences of these genes were determined. The gene clusteraccording to present invention involved in IPP biosynthesis of thenon-mevalonate pathway is involved in the biosynthesis of vitamin E andcarotenoids. Therefore, practical use plants with high contents ofvitamin E and carotenoids can be produced, by transforming plants by thegene cluster obtained in the present invention. More specifically, theHevea brasiliensis obtained by introducing the gene cluster of thepresent invention can be expected to produce high-quality rubber withimproved permanence.

1. An isolated protein consisting of the amino acid sequence of SEQ IDNO:
 8. 2. A gene encoding the protein according to claim
 1. 3. Anisolated gene consisting of the nucleotide sequence of nucleotide 150 to1085 of SEQ ID NO:
 7. 4. An isolated gene consisting of the nucleotidesequence of nucleotide 185 to 1117 of SEQ ID NO:
 7. 5. An isolated geneconsisting of the nucleotide sequence of nucleotide 1 to 1254 of SEQ IDNO:
 7. 6. An isolated gene consisting of the nucleotide sequence ofnucleotide 1 to 2069 of SEQ ID NO:
 7. 7. A transgenic plant, whereinproperties of the rubber produced from said plant is improved byintroducing the gene according to any one of claims 2 to 6 into saidplant.
 8. A method to improve the property of the rubber produced fromsaid plant, the method comprising introduction of the gene according toany one of claims 2 to 6 into said plant.